大鼠牙本质基质蛋白1单克隆抗体的制备、鉴定及其组织表达特异性研究
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摘要
牙本质非胶原蛋白(NCPs)在牙齿矿化过程中的作用一直是牙髓生物学的研究热点。应用组织学、生物化学和分子生物学等技术，从细胞水平、蛋白水平以至分子水平，已对其进行了大量的研究，使我们对牙本质矿化的过程和机理有了深入的理解。但目前的研究多集中于牙本质特异性蛋白，而对矿化组织特异性蛋白的研究相对较少。至于矿化组织特异性蛋白的确切生物学功能；它们之间以及它们与其他非胶原蛋白之间的相互作用；其在牙齿发育和骨组织发育中的作用：它们如何调节矿化过程，与牙本质发育不全症的关系等难题，仍有待我们作进一步深入研究。
牙本质基质蛋白1(dentin matrix protein 1 DMP1)是矿化组织特异性蛋白的重要成员之一，它富含天门冬氨酸、丝氨酸和谷氨酸，是一种分泌性磷酸化蛋白，与BAG-75有高度同源性，与骨桥素有部分同源性，它的组成介于骨酸性糖蛋白和牙齿磷酸化蛋白之间。研究表明：过表达DMP1基因的未分化间充质细胞可向成牙本质细胞样细胞分化，并可以使其在分化的早期阶段表达ALP、Cbfa1、BMP4，在分化的末期阶段表达BSP、OCN、OPN。而且随着DMP1表达量的增加，DMP2和DSP的表达量也增加。而用酪蛋白激酶Ⅱ抑制剂阻断DMP1磷酸化，DMP2、DSP、Cbfa1、OCN的表达未见明显变化。同时，过表达DMP1
京回军巨大攀双士学位论文
基因的成牙本质样细胞可以促进细胞内矿化小结的形成和增大。由此可推测，
体内 DMP在未分化间充质细胞向成牙本质细胞分化以及骨和牙齿硬组织形成
过程中起着非常重要的作用。
目前关于 DMP的研究集中于分子生物学结构和功能。由于缺乏良好的工
召 具，其在蛋白水平的表达和功能研究较少。为获得良好的研究工具，以揭示
DMPI蛋白在组织矿化过程中所起的作用，本实验组制备了抗大鼠DMPI的单克
隆抗体，并初步观察其生物学功能。
主要研究内容及结果如下：
一．大鼠牙本质基质蛋白1对体外培养的人牙髓细胞增殖和碱性磷酸酶活性
的影响
利用MTT法研究牙髓细胞的细胞增殖情况，并研究DMPI对牙髓细胞内碱性
磷酸酶活性的影响。DMPI的浓度为0．05ty／ml、0．fly／ml、0．sug／ml、互W／ml
和5％／ml。MTT实验刺激时间分别为id、3d、sd，结果显示：id组与对照组比
较无明显差异；3d和sd的sty砌组可促进人牙髓细胞增殖（P＜0．05），3d和
sd的其它各浓度组则与对照组无明显差异（P＞0．05）。ALP活性测定刺激时间
分别为3d、sd、7d，结果显示：细胞培养3d时，仅SPg砌1组可促进人牙髓细
胞从P活性（P（．05）；培养sd时fly／ml和sty／。l均可促进人牙髓细胞队P活
性（K0．05）；培养7d时促进作用进一步加强（k0．05）；sd组和7d组之间
无明显差异（P＞0．05）。结果提示：DMPI对牙髓细胞ALP活性的促进作用呈浓
度依赖性；高浓度DMP可以促进人牙髓细胞ALP活性，随着时间的延长，促进
ALP活性的作用也增强。由此表明DMPI在一定浓度下可促进牙髓细胞的增殖和
分化。
二．抗大鼠DMPI单克隆抗体的制备和鉴定
以大鼠重组DMP为抗原，免疫Balb／c ，J’鼠，通过B淋巴细胞杂交瘤技术，
即用B淋巴细胞与骨髓瘤细胞SP2／0进行细胞融合，利用间接ELISA法筛选
D ePeParo刃ent Of OPeOPerat。Dent。卿andblaoaontics 厂力山YU
3
章旧罩区大学呵士学位论文
阳性克隆，制备抗大鼠 DMP的单克隆抗体（MCAB），并对其性质进行鉴定。
结果：共获得2株单克隆抗体细胞株。dot hi。t结果显示2株抗体均与DMPI
和牙齿组织总蛋白能够反应，而与脑组织、肝脏、肾脏组织总蛋白无反应，
说明2株抗体均具有较好的特异性。从亲和常数可知ZH10的亲和力大于2C10
的亲和力。本实验首次获得了特异性较好、效价较高的大鼠 DMP单克隆抗体，
4
为进一步研究DMPI的确切生理功能提供了良好的工具。
三．牙本质基质蛋白l在大鼠不同组织中的表达研究
选用处于不同发育时期SD大鼠牙胚和牙齿；不同发育时期成年鼠的股骨
以及成年大鼠肝脏、肾脏和脑组织。采用免疫组织化学的方法，对 DMP在大
鼠不同组织中的表达进行研究。结果显示：D惭 在出生后3d大鼠磨牙和切牙
（牙本质基质分泌期）的成牙本质细胞胞浆顶端阳性表达，表明 DMP可能以
分泌颗粒的形式集聚于成牙本质细胞胞浆顶端，最终沿成牙本质细胞突分泌
Bone and dentin are mineralized tissues that resemble each other in the composition and mechanism of formation. In addition to a predominant collagenous matrix, bone and dentin contain non-collagenous proteins (NCPs). Since these NCPs are very acidic and are secreted into the extracellular matrix (ECM) during the formation and mineralization of these tissues, it is generally accepted that they play key biological roles in regulating the size and shape of the mineral crystal. Sialic acid-rich proteins, a category of mineralized tissue NCPs, include osteopontin(OPN), bone sialoprotein (BSP), bone acid glycoprotein-75 (BAG-75), dentin matrix protein 1(DMP1), and dentin sialoprotein (DSP). The precise functions of all these proteins are unknown.
DMP1 is an acidic protein that was first cloned from the mineralized dentin matrix. A unique feature of DMP1 is that it is highly hydrophilic. DMP1 is rich with glutamic acid, aspartic acid, and serines. The serine residues are embedded in acidic sequences that make them good substrates for phosphorylation by casein kinase I and II. It could be postulated that DMP1, because of its high acidic nature, strong binding power to calcium, could initiate the nucleation process and turn on the entire cascade of regulated hydroxyapatite crystal growth. Although the precise function of DMP1 is yet not to be determined, in situ hybridization experiments have demonstrated that it is localized in both osteoblasts and odontoblasts. At the amino acid level, DMP1 has a single RGD domain, which was shown recently to be functional in cell attachment. A detailed understanding of the physiological function of DMP1 is still lacking, even though this protein has been well characterized. The main contents and results of the study are presented as follows: 1. The effects of DMP1 on the growth and alkaline phosphatase activity
of cultured human dental pulp cells in vitro
The effects of DMP1 on the growth and alkaline phosphatase (ALP) activity of cultured human dental pulp cells were determined by MTT colorimetric assay and enzyme dynamical method. The concentrations of DMP1 added into culture medium were 0. 05M-g/ml, 0. iMg/ml, 0. SM-g/ml, lUg/ml and 5Mg/ml, which absence of DMP1 in culture medium was used as control. In MTT, Id, 3d and 5d were observed, the results shew that groups of Id there are no significant difference compared with control.
5Mg/ral DMP1 in 3d and 5d stimulated the cell proliferation compared with control (p<0. 05), no diferrences were observed between 5Mg/ml DMP1 in groups of 3d and 5d. Other groups of DMP1 in 3d and 5d had no significant difference(p>0. 05)compared with control. Determining time of ALP activity were 3d, 5d and 7d. Results shew that among 3d groups, ALP activity was increased (p<0. 05) by DMP1 at 5Hg/ml compared with control. Among 5d groups, ALP activity was increased (p<0. 05) by DMP1 at iP-g/ml and 5^g/ml. Among 7d groups, the effect of DMP1 still increased. These results indicated that DMP1 could stimulate the growth of cultured dental pulp cell, and high concentrations of DMP1 could increase ALP activity of dental pulp cell. 2. Preparation and identification of monoclonal antibodies against rat
recombinant DMP1
In order to prepare monoclonal antibodies (McABs) specifically against dentin matrix protein 1 (DMP1), we immunized Balb/c mice with rat recombined DMP1. Then McABs were produced by hybridomas derived from sp2/0 myeloma cells and spleen cells of immunized Balb/c mice. Results: Two hybridome cell lines, which stably secreted McABs against DMP1 were produced. Both McABs belonged to subclasses of IgGl. The results of dot hot shew that two McABs could specifically react with rat recombinant DMP1 and all dental proteins of rat, but could not react with any protein of rat brain, liver and kidney. The results indicated that two McABs obtained by hybridome technique had high affinity and were specific with DMP1. So far as we know, this was the first time about McABs of DMP1 reported. They could be of some help in the study
牙本质基质蛋白1(dentin matrix protein 1 DMP1)是矿化组织特异性蛋白的重要成员之一，它富含天门冬氨酸、丝氨酸和谷氨酸，是一种分泌性磷酸化蛋白，与BAG-75有高度同源性，与骨桥素有部分同源性，它的组成介于骨酸性糖蛋白和牙齿磷酸化蛋白之间。研究表明：过表达DMP1基因的未分化间充质细胞可向成牙本质细胞样细胞分化，并可以使其在分化的早期阶段表达ALP、Cbfa1、BMP4，在分化的末期阶段表达BSP、OCN、OPN。而且随着DMP1表达量的增加，DMP2和DSP的表达量也增加。而用酪蛋白激酶Ⅱ抑制剂阻断DMP1磷酸化，DMP2、DSP、Cbfa1、OCN的表达未见明显变化。同时，过表达DMP1
京回军巨大攀双士学位论文
基因的成牙本质样细胞可以促进细胞内矿化小结的形成和增大。由此可推测，
体内 DMP在未分化间充质细胞向成牙本质细胞分化以及骨和牙齿硬组织形成
过程中起着非常重要的作用。
目前关于 DMP的研究集中于分子生物学结构和功能。由于缺乏良好的工
召 具，其在蛋白水平的表达和功能研究较少。为获得良好的研究工具，以揭示
DMPI蛋白在组织矿化过程中所起的作用，本实验组制备了抗大鼠DMPI的单克
隆抗体，并初步观察其生物学功能。
主要研究内容及结果如下：
一．大鼠牙本质基质蛋白1对体外培养的人牙髓细胞增殖和碱性磷酸酶活性
的影响
利用MTT法研究牙髓细胞的细胞增殖情况，并研究DMPI对牙髓细胞内碱性
磷酸酶活性的影响。DMPI的浓度为0．05ty／ml、0．fly／ml、0．sug／ml、互W／ml
和5％／ml。MTT实验刺激时间分别为id、3d、sd，结果显示：id组与对照组比
较无明显差异；3d和sd的sty砌组可促进人牙髓细胞增殖（P＜0．05），3d和
sd的其它各浓度组则与对照组无明显差异（P＞0．05）。ALP活性测定刺激时间
分别为3d、sd、7d，结果显示：细胞培养3d时，仅SPg砌1组可促进人牙髓细
胞从P活性（P（．05）；培养sd时fly／ml和sty／。l均可促进人牙髓细胞队P活
性（K0．05）；培养7d时促进作用进一步加强（k0．05）；sd组和7d组之间
无明显差异（P＞0．05）。结果提示：DMPI对牙髓细胞ALP活性的促进作用呈浓
度依赖性；高浓度DMP可以促进人牙髓细胞ALP活性，随着时间的延长，促进
ALP活性的作用也增强。由此表明DMPI在一定浓度下可促进牙髓细胞的增殖和
分化。
二．抗大鼠DMPI单克隆抗体的制备和鉴定
以大鼠重组DMP为抗原，免疫Balb／c ，J’鼠，通过B淋巴细胞杂交瘤技术，
即用B淋巴细胞与骨髓瘤细胞SP2／0进行细胞融合，利用间接ELISA法筛选
D ePeParo刃ent Of OPeOPerat。Dent。卿andblaoaontics 厂力山YU
3
章旧罩区大学呵士学位论文
阳性克隆，制备抗大鼠 DMP的单克隆抗体（MCAB），并对其性质进行鉴定。
结果：共获得2株单克隆抗体细胞株。dot hi。t结果显示2株抗体均与DMPI
和牙齿组织总蛋白能够反应，而与脑组织、肝脏、肾脏组织总蛋白无反应，
说明2株抗体均具有较好的特异性。从亲和常数可知ZH10的亲和力大于2C10
的亲和力。本实验首次获得了特异性较好、效价较高的大鼠 DMP单克隆抗体，
4
为进一步研究DMPI的确切生理功能提供了良好的工具。
三．牙本质基质蛋白l在大鼠不同组织中的表达研究
选用处于不同发育时期SD大鼠牙胚和牙齿；不同发育时期成年鼠的股骨
以及成年大鼠肝脏、肾脏和脑组织。采用免疫组织化学的方法，对 DMP在大
鼠不同组织中的表达进行研究。结果显示：D惭 在出生后3d大鼠磨牙和切牙
（牙本质基质分泌期）的成牙本质细胞胞浆顶端阳性表达，表明 DMP可能以
分泌颗粒的形式集聚于成牙本质细胞胞浆顶端，最终沿成牙本质细胞突分泌
Bone and dentin are mineralized tissues that resemble each other in the composition and mechanism of formation. In addition to a predominant collagenous matrix, bone and dentin contain non-collagenous proteins (NCPs). Since these NCPs are very acidic and are secreted into the extracellular matrix (ECM) during the formation and mineralization of these tissues, it is generally accepted that they play key biological roles in regulating the size and shape of the mineral crystal. Sialic acid-rich proteins, a category of mineralized tissue NCPs, include osteopontin(OPN), bone sialoprotein (BSP), bone acid glycoprotein-75 (BAG-75), dentin matrix protein 1(DMP1), and dentin sialoprotein (DSP). The precise functions of all these proteins are unknown.
DMP1 is an acidic protein that was first cloned from the mineralized dentin matrix. A unique feature of DMP1 is that it is highly hydrophilic. DMP1 is rich with glutamic acid, aspartic acid, and serines. The serine residues are embedded in acidic sequences that make them good substrates for phosphorylation by casein kinase I and II. It could be postulated that DMP1, because of its high acidic nature, strong binding power to calcium, could initiate the nucleation process and turn on the entire cascade of regulated hydroxyapatite crystal growth. Although the precise function of DMP1 is yet not to be determined, in situ hybridization experiments have demonstrated that it is localized in both osteoblasts and odontoblasts. At the amino acid level, DMP1 has a single RGD domain, which was shown recently to be functional in cell attachment. A detailed understanding of the physiological function of DMP1 is still lacking, even though this protein has been well characterized. The main contents and results of the study are presented as follows: 1. The effects of DMP1 on the growth and alkaline phosphatase activity
of cultured human dental pulp cells in vitro
The effects of DMP1 on the growth and alkaline phosphatase (ALP) activity of cultured human dental pulp cells were determined by MTT colorimetric assay and enzyme dynamical method. The concentrations of DMP1 added into culture medium were 0. 05M-g/ml, 0. iMg/ml, 0. SM-g/ml, lUg/ml and 5Mg/ml, which absence of DMP1 in culture medium was used as control. In MTT, Id, 3d and 5d were observed, the results shew that groups of Id there are no significant difference compared with control.
5Mg/ral DMP1 in 3d and 5d stimulated the cell proliferation compared with control (p<0. 05), no diferrences were observed between 5Mg/ml DMP1 in groups of 3d and 5d. Other groups of DMP1 in 3d and 5d had no significant difference(p>0. 05)compared with control. Determining time of ALP activity were 3d, 5d and 7d. Results shew that among 3d groups, ALP activity was increased (p<0. 05) by DMP1 at 5Hg/ml compared with control. Among 5d groups, ALP activity was increased (p<0. 05) by DMP1 at iP-g/ml and 5^g/ml. Among 7d groups, the effect of DMP1 still increased. These results indicated that DMP1 could stimulate the growth of cultured dental pulp cell, and high concentrations of DMP1 could increase ALP activity of dental pulp cell. 2. Preparation and identification of monoclonal antibodies against rat
recombinant DMP1
In order to prepare monoclonal antibodies (McABs) specifically against dentin matrix protein 1 (DMP1), we immunized Balb/c mice with rat recombined DMP1. Then McABs were produced by hybridomas derived from sp2/0 myeloma cells and spleen cells of immunized Balb/c mice. Results: Two hybridome cell lines, which stably secreted McABs against DMP1 were produced. Both McABs belonged to subclasses of IgGl. The results of dot hot shew that two McABs could specifically react with rat recombinant DMP1 and all dental proteins of rat, but could not react with any protein of rat brain, liver and kidney. The results indicated that two McABs obtained by hybridome technique had high affinity and were specific with DMP1. So far as we know, this was the first time about McABs of DMP1 reported. They could be of some help in the study
引文
1． Butler WT, and Ritchie, HH. The nature and functional significance of dentin extracellular matrix protein. Int J Dev Biol. 1995;39:169
2． Linde A, Goldberg M. Dentinogenesis. Critical Rewiew in Oral Biology and Medicine. 1993,4(5) :679-728
3． Septier D, Torres-Quintana MA, Menashi S et al. Inositol hexasulphate, a casein kinase inhibitor, alters the distribution of dentin matrix protein 1 in cultured embryonic mouse tooth germs. Eur J Oral Sci 2001; 109:198
4． Goldberg M, D Septier, F Escaig-Haye. Glycoconjugates in denitnogenesis and dentin. Progr Histochem Cytochem. 1987 pp1-113． 17: No 2, Fischer Verlag, Stuttgart.
5． Nagata K, Huang YH, Ohsaki Y et al Demonstration of type Ⅲ collagen in the dentin of mice. Matrix. 1992; 12:448-455
6． Cournil I, C P Leblond, J Pomponio et al Immunohistochemical localization of procollangens Ⅰ Ⅲ and Ⅳ antigenicity in the rat incisor tooth by the indirect peroxidase-antiperoxidase method. J Histochem Cytochem.1979;27:1059-1069
7． Dodd C M, D J Carmichael. The collagenous matrix of bovine predentine. Biolchim Biophys Acta. 1979;577:117-124
8． Munksgaard E C, D Moe. Types of collagen in an extract of odontoblasts and dentin from developing bovine teeth. Archs Oral Biol. 1980;25:485-489
9． Eyre D. Collagen crosslinks amino acids. Methods Enzymol. 1987; 144:115-139
10． Niyibizi C, and Eyre DR.Structural characteristics of cross-lining sites in type Ⅴ collagen of bone. Chain specificities and heterotypic links to type Ⅰ collagen. Eur J Biolchem. 1994;224:943
11． Birk DE, Fitch JM, Barbiaz JP et al. Collagen fibrogenesis in vitro interactions of type Ⅰ and V collagen regulate fibril diameter. J Cell Sci. 1990;95: 649
12． Bultler WT. Dentin matrix proteins and dentinogenesis. Connect Tissue Res. 1995;3 (1-3) :59-65
13． Ritchie HH, Hou H, Veis A, Butler WT. Cloning and sequence determination of rat dentin sialoprotein,a novel dentin protein. J Biol Chem. 1994,269(5) :3698-702．
14． Ritchie HH, Pinero GJ, Hou H, Butler WT. Molecular analysis of rat dentin sialoprotein. Connect Tissue Res. 1995;33(1-3) :73-79
15． D'souza. RN, Bronckers.AL, Happonen.RP. Developmental expression of a 53kD dentin
sialoprotein in rat tooth organs. JHistochem Cytochem.1992; 40(3) :359-366
16． Bronkers AL, D'zousa RN, Butler WT. Dentin sialoprotein: biosythesis and developmental appearance in rat tooth germs in comparison with amelogenins.osteocalcin and collagen type I . Cell Tissue Res 1993, 273(2) :237-247
17． Ronckers.AL, Dsouza.RN, Butler.W.T., etal. Dentin sialoproein: biosynthesis and developmental appearance in rat tooth germs in comparison wit amelogenins,osteocalci and collegen type I . Cell Tissue Res. 1993; 272(2) :237-247
18． Wada Y, Fujisawa Y, Nodasaka Y, and Kuboki Y. Electrophoretic gel of dentin matrix protein as diffusion media or in vitro mineralization. J Dent Res.1996; 75(6) :1381-1387
19． Osamu Nakanura, Eiichi Gohda, Masayuki ozawa, et al. Immunohistochemical studies with a monoclone antibody on the distribution of phosphophoryn in predentin and dentin. Calcif Tissue Int. 1985; 37:491-500
20． Linde A. Difference between non-collagenous protein content of rat incisor and permenent bovine dentin. Scznd J Dent Res. 1988; 96:188
21． 赵大为．牙本质磷蛋白．牙体牙髓牙周病学杂志，1995；5(3) ：187
22． .Vries IGD, Quatier E, Steireghem V. Characterization and immunocytochemical localization of dentin phosphoprotein in rat and bovine teeth. Archs Oral Biol. 1986; 31: 574
23． Chang SR, Chiego D Jr, Clarkson BH. Characterization and identification of a human dentin phosphophoryn. Calcif Tissue Int. 1996 Sep;59(3) : 149-53
24． Gu K, Chang SR, Slaven MS et al. Human dentin phosphophoryn nucleotide and amino acid sequence. Eur J Oral Sci. 1998 Dec; 106(6) : 1043-7
25． MacDougall M, Simmons D, Luan X et al. Dentin phosphoprotein and dentin sialoprotein are cleavage products expressed from a single transcript coded by a gene on human chromosome 4． J Biol Chem 1997 Jan 10;272(2) :835-42
26． Linde A. Dentin and dentinogenesis, CRC Press.Boca Raton, FL. 1984,Vol 2:55-92．
27． Linde A, Bhown M, Butler WT. Noncollagenous proteins of dentin. A re-examination of proteins from rat incisor dentin utilizing techniques to avoid artifacts. J Biol Chem. 1980; 255(12) :5931-42
28． Richardson WS, Munksgaard EC, Butler WT. Rat incisor phosphoprotein. The nature of the phosphate and quantitation of the phosphoserine. J Biol Chem 1978,253(22) :8042-46
29． Veis A, Perry A. The phosphoprotein of the dentin matrix. Biochemistry 1967, 6(8) : 2409-16
30． Jonsson MS, Fredricsson S, Jontell M, Linde A. Isoelectric focusing of the phosphoprotein of rat-incisor dentin in ampholine and acid pH gradients. Evidence for carrier ampholyte-protein complexes. J Chromatogr 1978, 157:235-242
31． Lee SL, Veis A, Glonek T. Dentin phosphoprotein: an extracellular calcium-binding protein. Biochemistry 1977,16:2971-2979
32． Butler WT, Bhown M, DiMuzio MT, Cothran WC, Linde A. Multiple forms of rat dentin phosphoproteins. Arch Biochem Biophys. 1983; 225(1) : 178-86．
33． Ritchie HH, Wang L-H. Sequence determination of an extremely acidic rat dentin phosphoprotein. J Biol Chem 1996;271:21695-21698
34． George A, Bannon L, Sabsay B, Dillon J, Malone J, Veis A, Jenkins N, Gilbert D, Copeland N. The carboxylterminal domain of phosphophoryn contains unique extended amino acid repeat sequences forming ordered carboxyl-phosphate interaction ridges that may be essential in the biomineralization process. J Biol Chem 1996;271:32869-32873
35． Macdougall.M, Simmons.D, xianghong Luan, etal. Dentin phosphoprotein and dentin sialoprotein are cleavage products expressed from a single transcript coded by a gene on human chromosome 4． J Biolo Chem , 1997; 272(2) : 835-842
36． Nawrot CF, Campbell DJ, Schroeder JK, Van Valkenburg M. Dental phosphoprotein-induced formation of hydroxyapatite during in vitro synthesis of amorphous calcium phosphate. Bioche Chem 1976,15:3445-3449
37． Fujisawa R, Takagi T, Kuboki Y, Sasaki S. Systematic purification of free and matrix-bound phosphophoryns of bovine dentin: Presence of matrix-bound phosphophoryns as a distinct molecular entity. Calcif Tissue Int 1984,36:239-242
38． Hohling HJ, Arnold S, Barckhaus RH, et al. Structural relationship between the primary crystal formations and the matrix macromolecules in different hard tissues. Discussion of a general principle. Connect Tissue Res, 1995; 33(1-3) :171-178
39． Boskey AL. The role of extracellular matrix components in dentin mineralization. Crit Rev Oral Biol Med, 1991;2(3) :369-387
40． Boskey A, Muresa S, Doty S.Sabsay B, Veis A. Concentration dependant effects of dentin phosphophoryn in the regulation of in vitro hydroxyapatite formation and growth. Bone Miner 1990; 11:55-6
41． Hunter G, Hauschka P, Poole A, Rosenberg L, Goldberg H.Nucleation and inhibition of
hydroxyapatite formation by mineralized tissue proteins. Biochem J 1996;317:5964
42. Butler WT. Dentin specific proteins. Methods Enzymol 1987;145:290-303
43. Feng JQ, Luan X, MacDougall M et al. Genomic organization, chromosomal mapping, and promoter analysis of the mouse dentin sialophosphoprotein (Dspp) gene, which codes for both dentin sialoprotein and dentin phosphoprotein. J Biol Chem 1998 Apr 17; 273(16): 9457-64
44. Gu K, Chang S, Ritchie HH et al. Molecular cloning of a human dentin sialophosphoprotein gene. Eur J Oral Sci 2000 Feb; 108(1):35-42
45. George A, Srinivasan RThotakura SR, Liu K, Veis A. Rat dentin matrix protein 3 is a compound protein of rat dentin sialoprotein and phosphophoryn. Connect Tissue Res.1999; 40(1):49-57.
46. Bleicher F, Couble ML, Farges JC, et al. Senquential expression of matrix protein genes in developing rat teeth. Matix Biol, 1999: 18(2): 133-143
47. Begue-Kim C, Krebsbach PH, Bartlett JD, Butler WT. Dentin sialoprotein, dentin phosphoprotein, enamelysin and ameloblastin: tooth-specific molecules that are distinctively expressed during murine dental differentiation. Eur J Oral Sci 1998,106:963-970
48. Begue-Kirn C, Ruch JV, Ridall AL, Butler WT. Comparative analysis of mouse DSP and DPP expression in odontoblasts, preameioblasts, and experimentally induced odontoblast-like cells. Eur J Oral Sci 1998; 106(suppl):254-259
49．江卫民．小鼠牙本质涎磷蛋白cDNA克隆、表达、纯化、抗体制备用其组织表达特异性研究。第四军医大学博士学位论文。1999年
50．张蓉 肖明振 赵守亮等 小鼠牙胚、软骨组织中牙持质涎磷蛋白基因的克隆 牙体牙髓牙周病学杂志 2001；11(4)：213-
51. Rahemtulla F C, W Prince, W T Butler. Isolation and partial characterization of proteoglycans from rat insicors. Biochem J. 1984;218:877-885
52. Goldberg M, F Escaig. The appearance in TEM of proteoglycan in predentine is fixation dependent. J Microsc. 1984a; 134:161-167
53. Chardin H, I londono, M Goldberg. Visualization of glycosaminoglycans in rat incisor extracellular matrixusing a hyaluronidase-gold complex. Histochem J. 1990;22:588-594
54. Obrink B. The influence of glycosaminoglycans on the formation of fibers from monomeric tropocollagen in vitro. Eur J Biochem. 1973;34:129-137
55． Chen J, Shapiro HS, Sodek. Development expression of bone sialoprotein mRNA in rat mineralized tissues. J Bone Miner Res. 1992; 7:987
56． Hunter GK, Goldberg HA. Modulation of crystal formation by bone phosphoprotein: role of gultamic acid-rich sequences in the nucleation of hydroxyapatite by bone sailoprotein. Biochem J 1994; 302: 175
57． Gorier De Vries, L D Coomans, E Wisse. Ultrastructural localization of osteocalcin in rat tooth germs by immunogold staining. Histochmistry; 1988a:89:509-514
58． Goiter De Vries, L D Coomans, E Wisse. Immunocytochemical localization of osteocalcin in human and bovine teeth. Calcif Tissue Int. 1988b; 43:128-130
59． Butler WT. The nature and significance of osteopontin. Connect Tissue Res;1989:23:123
60． Young MF, Kerr, Termine JD et al. cDNA clong, mRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin(OPN). Genomics. 1990;7:491
61． Gorski JP and Shimizu K. Isolation of new phosphorylated glycoprotein from mineralized phase of bone that exhibits limited homology toadhesive protein ostepontin. J Biol Chem. 1988;263:15938
62． Gorski JP, Griffin D, Dudley G, et al. Bone acidic glycoprotein-75 (BAG-75) is a major synthetic product of osteoblastic cells and localized as 75 and/or 50-kDa forms in mineralized phases of bone and growth plate and in serum. J Biol Chem.1990;265:14956
63． Gorski JP, Kremer EA, Chen Y,et al. Bone acidic glycoprotein-75 (BAG-75) self-associates to form macromolecular complexes in vitro and in vivo with the potential to sequester phosphateions. J Cell Biochem .1997; 64:547
64． George A, Sabasy B, Simonian PAL et al. Characterization of a novel dentin matrix phosphoprotein. JBiol Chem 1993,268: 12642
65． George A, Gui J, Jenkins NA et al. In situ localization and chromosomal mapping of the AG1 (Dmpl) gene. J Histochem Cytochem 1994 Dec;42(12) : 1527-31
66． George A, Silberstein R, Veis A et al In situ hybridization shows DMP1 to be a developmentally regulated dentin-specific protein produced by mature odontblasts. Connect Rissue Res 1995 33(1-3) : 389-94
67． Thotakura SR, Karthikeyan N, George A et al. Cloning and characterization of rat dentin matrix protein 1 (DMP1) gene and its 5'-upstream region. J Biol Chem 2000 Apr 7;275(14) : 10272-7
68． Hirst KL, Ibaraki-O'Connor, Young MF et al. Cloning and expession analysis of the
bovine matrix acidic phosphoprotein gene. J Dent Res 1997,76;(3) : 754-760
69． Hirst KL, Simmons D, Feng J et al. Elucidation of the sequence and the genomic organization of tne human dentin matrix acdic phosphoprotein 1(DMP1) gene:exlusion of the locus from a causative role in the pathgenesis of dentinogenesis imperfecta type Ⅱ. Genomics 1997, 42;38-45
70． MacDougall M, Gu TT, Luan X et al. Identification of a novel isoform of mouse dentin matrix protein 1: spatial expression in mineralized tissues. J Bone Miner Res 1998 Mar;13(3) :422-31
71． Srinivasan R, Chen B, Gorski JP, George A et al. Recombinant expression and characterization of dentin matrix protein 1． Connect Tissue Res 1999;40(4) :251-8
72． Qin C, Brunn JC, George A et al. A comparative study of sialic acid-rich proteins in rat bone and dentin. Eur J Oral Sci. 2001; 109: 133-141
73． D'Souza RN, Cavender A, MacDougall M et al. Gene expression patterns of murine dentin matrix protein 1 (Dmp1) and dentin sialophosphoprotein (DSPP) suggest distinct developmental functions in vivo. J Bone Miner Res 1997 Dec;12(12) :2040-9
74． Septier D, Torres-Quintana M A, Menashi S et al Inositol hexasulphate, a casein kinase inhibitor, alters the distribution of dentin matrix protin 1 in cultured embryonic mouse tooth grms. Eur J Oral Sci 2001; 109:198-293
75． Kulkarni GV, Chen B, George A et al. Promotion of selective cell attachment by the RGD sequence in dentine matrix protein 1． Arch Oral Biol 2000 Jun;45(6) :475-84
76． Veis, A. (1996) in Extracellular Matrix, ed. Comper, W. D. (Harwood, London), pp. 41-76．
77． Narayanan K, Srinivas R, Ramachandran A, George A. Differentiation of embryonic mesenchymal cells to odontoblast-like cells by overexpression of dentin matrix protein 1 Proc. Natl. Acad. Sci. USA, Vol. 98, Issue 8, 4516-4521, April 10, 200
78． Franch D, Fischberg E, Buhl S et al. The production of more useful monoclonal antibodies. Immunol Today 1986;7:344
79． Nilsson BO and Larsson A. Intrasplenic immunization with minute amounts of antigen. Immunol Today 1990; 11:10
80． Coyle PV, Wyatt D, Mccaughey C et al. A simple standardised protocol for the production of monoclonal antibodies against viral and bacterial antigens.J Immunol Methods 1992;153:81
81. Hengartner A, Luzzati AL and Schreier M. Fusion of in vitro immunized lymphoid cells with X63Ag8. Curt Top Microbiol Immunol 1978; 81:92
82．金伯泉主编．细胞和分子免疫学．西安，世界图书出版公司1995年：118
83. Lo MS, Tsong TY,Conrad MK et al. Monoclonal antibody production by receptor-mediated electrically induced cell fusion. Nature 1984; 310:792
84. Vitetta ES and Uhr JW. Monoclonal an tibodies as agonists:an expanded role for their use in cancer therapy. Cancer Res 1994;54:5301
85. Riechmann L and Clark M. Reshaping human antibodies for therapy. Nature 1988;332:323
86. Chiswell DJ and McCafferty J. Phage antibodies: will now "Coliclonal" antibodies replace monoclonal antibodies? TIBTECH 1992; 10:80
87. Maclennan I. The center of hypermutation. Nature 1991;354:352
88. Ward ES, Gussow D, Griffiths AD et al. Binding activities of a repertoire of single Ig variable domain secreted from E. coli. Nature 1989;341:544
89. Pluckthun A.Mono-and bivalent antibody fragment produced in E. coli:engineering, folding and antigen binding. Immunol Rev 1992; 130:151
90. Barbas CF, Bain JD, Hoekstra DM et al.Semisynthethetic combinatorial antibody libraries:chemical solution to the diversity problem. PNAS 1992;89:4457
91. Tinter G, and Griffithe AD. Making antibodies by phage display technology. Ann Rev Immunol 1994;12:433
92．陈竞华，王琰，刘群英等。人源性噬菌体抗体库的构建及HbsAg单抗的筛选。中华微生物学和免疫学杂志，1995；15：158
93. Ali SY, Anderson HC,Sajdera SW.Enzymic and electron-microscopic analysis of extracellular matrix vesicle assiciated calcification in carting. Biochem J. 1997,132;56
94．陈筠．牙髓碱性磷酸酶的提纯、cDNA克隆和氢氧化钙对其活性、表达的影响：[学位论文]。第四军医大学口腔医学院，1991．9
95．郝建军 激素和生长因子与牙乳头细胞，牙髓细胞，牙胚分化和矿化关系的体外实验研究 第四军医大学 博士学位论文 1997 5
96. Hao J, Shi S,Niu Z et al.Mineralized nodule formation by human dental papilla cells in culture. Eur J Oral Sci 1997 Aug; 105(4): 318-24
97. Goseki M, Oida S, Nifuji A et al Properties of alkaline phosphatase of the human dental pulp. J Dent Res. 1990, 69:909
98．徐志凯 实用单克隆抗体技术第1版 陕西科学技术出版社 1992
99. Laboux O, Menanteau J, Pelhate S et al. Development of a monoclonai antibody against dentin phosphophoryn: a tool to study odontoblastic activity. Hybridoma. 1994; 13(2):143-146
100．陶义询 主编 免疫学的免疫学检验 第二版 人民卫生出版社 1997
101. J.D Beatty, Barbara G, William G et al. Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. Journal of immunological methods. 1987;100(1987): 173-179
102. Kulkarni GV, Jee SW, Thotakura S, et al Differential expresstion of DMP1,2 and 3transcripts in carly stages of tooth development. In Goldberg M, Robinson C, Boskey AL, eds. Proceeding 6th International conference on the Chemistry and Biology of Minerallized Tissues. Rosemont, IL:American Academy of Orthopaedic Surgeons. 2000; 161-195
2． Linde A, Goldberg M. Dentinogenesis. Critical Rewiew in Oral Biology and Medicine. 1993,4(5) :679-728
3． Septier D, Torres-Quintana MA, Menashi S et al. Inositol hexasulphate, a casein kinase inhibitor, alters the distribution of dentin matrix protein 1 in cultured embryonic mouse tooth germs. Eur J Oral Sci 2001; 109:198
4． Goldberg M, D Septier, F Escaig-Haye. Glycoconjugates in denitnogenesis and dentin. Progr Histochem Cytochem. 1987 pp1-113． 17: No 2, Fischer Verlag, Stuttgart.
5． Nagata K, Huang YH, Ohsaki Y et al Demonstration of type Ⅲ collagen in the dentin of mice. Matrix. 1992; 12:448-455
6． Cournil I, C P Leblond, J Pomponio et al Immunohistochemical localization of procollangens Ⅰ Ⅲ and Ⅳ antigenicity in the rat incisor tooth by the indirect peroxidase-antiperoxidase method. J Histochem Cytochem.1979;27:1059-1069
7． Dodd C M, D J Carmichael. The collagenous matrix of bovine predentine. Biolchim Biophys Acta. 1979;577:117-124
8． Munksgaard E C, D Moe. Types of collagen in an extract of odontoblasts and dentin from developing bovine teeth. Archs Oral Biol. 1980;25:485-489
9． Eyre D. Collagen crosslinks amino acids. Methods Enzymol. 1987; 144:115-139
10． Niyibizi C, and Eyre DR.Structural characteristics of cross-lining sites in type Ⅴ collagen of bone. Chain specificities and heterotypic links to type Ⅰ collagen. Eur J Biolchem. 1994;224:943
11． Birk DE, Fitch JM, Barbiaz JP et al. Collagen fibrogenesis in vitro interactions of type Ⅰ and V collagen regulate fibril diameter. J Cell Sci. 1990;95: 649
12． Bultler WT. Dentin matrix proteins and dentinogenesis. Connect Tissue Res. 1995;3 (1-3) :59-65
13． Ritchie HH, Hou H, Veis A, Butler WT. Cloning and sequence determination of rat dentin sialoprotein,a novel dentin protein. J Biol Chem. 1994,269(5) :3698-702．
14． Ritchie HH, Pinero GJ, Hou H, Butler WT. Molecular analysis of rat dentin sialoprotein. Connect Tissue Res. 1995;33(1-3) :73-79
15． D'souza. RN, Bronckers.AL, Happonen.RP. Developmental expression of a 53kD dentin
sialoprotein in rat tooth organs. JHistochem Cytochem.1992; 40(3) :359-366
16． Bronkers AL, D'zousa RN, Butler WT. Dentin sialoprotein: biosythesis and developmental appearance in rat tooth germs in comparison with amelogenins.osteocalcin and collagen type I . Cell Tissue Res 1993, 273(2) :237-247
17． Ronckers.AL, Dsouza.RN, Butler.W.T., etal. Dentin sialoproein: biosynthesis and developmental appearance in rat tooth germs in comparison wit amelogenins,osteocalci and collegen type I . Cell Tissue Res. 1993; 272(2) :237-247
18． Wada Y, Fujisawa Y, Nodasaka Y, and Kuboki Y. Electrophoretic gel of dentin matrix protein as diffusion media or in vitro mineralization. J Dent Res.1996; 75(6) :1381-1387
19． Osamu Nakanura, Eiichi Gohda, Masayuki ozawa, et al. Immunohistochemical studies with a monoclone antibody on the distribution of phosphophoryn in predentin and dentin. Calcif Tissue Int. 1985; 37:491-500
20． Linde A. Difference between non-collagenous protein content of rat incisor and permenent bovine dentin. Scznd J Dent Res. 1988; 96:188
21． 赵大为．牙本质磷蛋白．牙体牙髓牙周病学杂志，1995；5(3) ：187
22． .Vries IGD, Quatier E, Steireghem V. Characterization and immunocytochemical localization of dentin phosphoprotein in rat and bovine teeth. Archs Oral Biol. 1986; 31: 574
23． Chang SR, Chiego D Jr, Clarkson BH. Characterization and identification of a human dentin phosphophoryn. Calcif Tissue Int. 1996 Sep;59(3) : 149-53
24． Gu K, Chang SR, Slaven MS et al. Human dentin phosphophoryn nucleotide and amino acid sequence. Eur J Oral Sci. 1998 Dec; 106(6) : 1043-7
25． MacDougall M, Simmons D, Luan X et al. Dentin phosphoprotein and dentin sialoprotein are cleavage products expressed from a single transcript coded by a gene on human chromosome 4． J Biol Chem 1997 Jan 10;272(2) :835-42
26． Linde A. Dentin and dentinogenesis, CRC Press.Boca Raton, FL. 1984,Vol 2:55-92．
27． Linde A, Bhown M, Butler WT. Noncollagenous proteins of dentin. A re-examination of proteins from rat incisor dentin utilizing techniques to avoid artifacts. J Biol Chem. 1980; 255(12) :5931-42
28． Richardson WS, Munksgaard EC, Butler WT. Rat incisor phosphoprotein. The nature of the phosphate and quantitation of the phosphoserine. J Biol Chem 1978,253(22) :8042-46
29． Veis A, Perry A. The phosphoprotein of the dentin matrix. Biochemistry 1967, 6(8) : 2409-16
30． Jonsson MS, Fredricsson S, Jontell M, Linde A. Isoelectric focusing of the phosphoprotein of rat-incisor dentin in ampholine and acid pH gradients. Evidence for carrier ampholyte-protein complexes. J Chromatogr 1978, 157:235-242
31． Lee SL, Veis A, Glonek T. Dentin phosphoprotein: an extracellular calcium-binding protein. Biochemistry 1977,16:2971-2979
32． Butler WT, Bhown M, DiMuzio MT, Cothran WC, Linde A. Multiple forms of rat dentin phosphoproteins. Arch Biochem Biophys. 1983; 225(1) : 178-86．
33． Ritchie HH, Wang L-H. Sequence determination of an extremely acidic rat dentin phosphoprotein. J Biol Chem 1996;271:21695-21698
34． George A, Bannon L, Sabsay B, Dillon J, Malone J, Veis A, Jenkins N, Gilbert D, Copeland N. The carboxylterminal domain of phosphophoryn contains unique extended amino acid repeat sequences forming ordered carboxyl-phosphate interaction ridges that may be essential in the biomineralization process. J Biol Chem 1996;271:32869-32873
35． Macdougall.M, Simmons.D, xianghong Luan, etal. Dentin phosphoprotein and dentin sialoprotein are cleavage products expressed from a single transcript coded by a gene on human chromosome 4． J Biolo Chem , 1997; 272(2) : 835-842
36． Nawrot CF, Campbell DJ, Schroeder JK, Van Valkenburg M. Dental phosphoprotein-induced formation of hydroxyapatite during in vitro synthesis of amorphous calcium phosphate. Bioche Chem 1976,15:3445-3449
37． Fujisawa R, Takagi T, Kuboki Y, Sasaki S. Systematic purification of free and matrix-bound phosphophoryns of bovine dentin: Presence of matrix-bound phosphophoryns as a distinct molecular entity. Calcif Tissue Int 1984,36:239-242
38． Hohling HJ, Arnold S, Barckhaus RH, et al. Structural relationship between the primary crystal formations and the matrix macromolecules in different hard tissues. Discussion of a general principle. Connect Tissue Res, 1995; 33(1-3) :171-178
39． Boskey AL. The role of extracellular matrix components in dentin mineralization. Crit Rev Oral Biol Med, 1991;2(3) :369-387
40． Boskey A, Muresa S, Doty S.Sabsay B, Veis A. Concentration dependant effects of dentin phosphophoryn in the regulation of in vitro hydroxyapatite formation and growth. Bone Miner 1990; 11:55-6
41． Hunter G, Hauschka P, Poole A, Rosenberg L, Goldberg H.Nucleation and inhibition of
hydroxyapatite formation by mineralized tissue proteins. Biochem J 1996;317:5964
42. Butler WT. Dentin specific proteins. Methods Enzymol 1987;145:290-303
43. Feng JQ, Luan X, MacDougall M et al. Genomic organization, chromosomal mapping, and promoter analysis of the mouse dentin sialophosphoprotein (Dspp) gene, which codes for both dentin sialoprotein and dentin phosphoprotein. J Biol Chem 1998 Apr 17; 273(16): 9457-64
44. Gu K, Chang S, Ritchie HH et al. Molecular cloning of a human dentin sialophosphoprotein gene. Eur J Oral Sci 2000 Feb; 108(1):35-42
45. George A, Srinivasan RThotakura SR, Liu K, Veis A. Rat dentin matrix protein 3 is a compound protein of rat dentin sialoprotein and phosphophoryn. Connect Tissue Res.1999; 40(1):49-57.
46. Bleicher F, Couble ML, Farges JC, et al. Senquential expression of matrix protein genes in developing rat teeth. Matix Biol, 1999: 18(2): 133-143
47. Begue-Kim C, Krebsbach PH, Bartlett JD, Butler WT. Dentin sialoprotein, dentin phosphoprotein, enamelysin and ameloblastin: tooth-specific molecules that are distinctively expressed during murine dental differentiation. Eur J Oral Sci 1998,106:963-970
48. Begue-Kirn C, Ruch JV, Ridall AL, Butler WT. Comparative analysis of mouse DSP and DPP expression in odontoblasts, preameioblasts, and experimentally induced odontoblast-like cells. Eur J Oral Sci 1998; 106(suppl):254-259
49．江卫民．小鼠牙本质涎磷蛋白cDNA克隆、表达、纯化、抗体制备用其组织表达特异性研究。第四军医大学博士学位论文。1999年
50．张蓉 肖明振 赵守亮等 小鼠牙胚、软骨组织中牙持质涎磷蛋白基因的克隆 牙体牙髓牙周病学杂志 2001；11(4)：213-
51. Rahemtulla F C, W Prince, W T Butler. Isolation and partial characterization of proteoglycans from rat insicors. Biochem J. 1984;218:877-885
52. Goldberg M, F Escaig. The appearance in TEM of proteoglycan in predentine is fixation dependent. J Microsc. 1984a; 134:161-167
53. Chardin H, I londono, M Goldberg. Visualization of glycosaminoglycans in rat incisor extracellular matrixusing a hyaluronidase-gold complex. Histochem J. 1990;22:588-594
54. Obrink B. The influence of glycosaminoglycans on the formation of fibers from monomeric tropocollagen in vitro. Eur J Biochem. 1973;34:129-137
55． Chen J, Shapiro HS, Sodek. Development expression of bone sialoprotein mRNA in rat mineralized tissues. J Bone Miner Res. 1992; 7:987
56． Hunter GK, Goldberg HA. Modulation of crystal formation by bone phosphoprotein: role of gultamic acid-rich sequences in the nucleation of hydroxyapatite by bone sailoprotein. Biochem J 1994; 302: 175
57． Gorier De Vries, L D Coomans, E Wisse. Ultrastructural localization of osteocalcin in rat tooth germs by immunogold staining. Histochmistry; 1988a:89:509-514
58． Goiter De Vries, L D Coomans, E Wisse. Immunocytochemical localization of osteocalcin in human and bovine teeth. Calcif Tissue Int. 1988b; 43:128-130
59． Butler WT. The nature and significance of osteopontin. Connect Tissue Res;1989:23:123
60． Young MF, Kerr, Termine JD et al. cDNA clong, mRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin(OPN). Genomics. 1990;7:491
61． Gorski JP and Shimizu K. Isolation of new phosphorylated glycoprotein from mineralized phase of bone that exhibits limited homology toadhesive protein ostepontin. J Biol Chem. 1988;263:15938
62． Gorski JP, Griffin D, Dudley G, et al. Bone acidic glycoprotein-75 (BAG-75) is a major synthetic product of osteoblastic cells and localized as 75 and/or 50-kDa forms in mineralized phases of bone and growth plate and in serum. J Biol Chem.1990;265:14956
63． Gorski JP, Kremer EA, Chen Y,et al. Bone acidic glycoprotein-75 (BAG-75) self-associates to form macromolecular complexes in vitro and in vivo with the potential to sequester phosphateions. J Cell Biochem .1997; 64:547
64． George A, Sabasy B, Simonian PAL et al. Characterization of a novel dentin matrix phosphoprotein. JBiol Chem 1993,268: 12642
65． George A, Gui J, Jenkins NA et al. In situ localization and chromosomal mapping of the AG1 (Dmpl) gene. J Histochem Cytochem 1994 Dec;42(12) : 1527-31
66． George A, Silberstein R, Veis A et al In situ hybridization shows DMP1 to be a developmentally regulated dentin-specific protein produced by mature odontblasts. Connect Rissue Res 1995 33(1-3) : 389-94
67． Thotakura SR, Karthikeyan N, George A et al. Cloning and characterization of rat dentin matrix protein 1 (DMP1) gene and its 5'-upstream region. J Biol Chem 2000 Apr 7;275(14) : 10272-7
68． Hirst KL, Ibaraki-O'Connor, Young MF et al. Cloning and expession analysis of the
bovine matrix acidic phosphoprotein gene. J Dent Res 1997,76;(3) : 754-760
69． Hirst KL, Simmons D, Feng J et al. Elucidation of the sequence and the genomic organization of tne human dentin matrix acdic phosphoprotein 1(DMP1) gene:exlusion of the locus from a causative role in the pathgenesis of dentinogenesis imperfecta type Ⅱ. Genomics 1997, 42;38-45
70． MacDougall M, Gu TT, Luan X et al. Identification of a novel isoform of mouse dentin matrix protein 1: spatial expression in mineralized tissues. J Bone Miner Res 1998 Mar;13(3) :422-31
71． Srinivasan R, Chen B, Gorski JP, George A et al. Recombinant expression and characterization of dentin matrix protein 1． Connect Tissue Res 1999;40(4) :251-8
72． Qin C, Brunn JC, George A et al. A comparative study of sialic acid-rich proteins in rat bone and dentin. Eur J Oral Sci. 2001; 109: 133-141
73． D'Souza RN, Cavender A, MacDougall M et al. Gene expression patterns of murine dentin matrix protein 1 (Dmp1) and dentin sialophosphoprotein (DSPP) suggest distinct developmental functions in vivo. J Bone Miner Res 1997 Dec;12(12) :2040-9
74． Septier D, Torres-Quintana M A, Menashi S et al Inositol hexasulphate, a casein kinase inhibitor, alters the distribution of dentin matrix protin 1 in cultured embryonic mouse tooth grms. Eur J Oral Sci 2001; 109:198-293
75． Kulkarni GV, Chen B, George A et al. Promotion of selective cell attachment by the RGD sequence in dentine matrix protein 1． Arch Oral Biol 2000 Jun;45(6) :475-84
76． Veis, A. (1996) in Extracellular Matrix, ed. Comper, W. D. (Harwood, London), pp. 41-76．
77． Narayanan K, Srinivas R, Ramachandran A, George A. Differentiation of embryonic mesenchymal cells to odontoblast-like cells by overexpression of dentin matrix protein 1 Proc. Natl. Acad. Sci. USA, Vol. 98, Issue 8, 4516-4521, April 10, 200
78． Franch D, Fischberg E, Buhl S et al. The production of more useful monoclonal antibodies. Immunol Today 1986;7:344
79． Nilsson BO and Larsson A. Intrasplenic immunization with minute amounts of antigen. Immunol Today 1990; 11:10
80． Coyle PV, Wyatt D, Mccaughey C et al. A simple standardised protocol for the production of monoclonal antibodies against viral and bacterial antigens.J Immunol Methods 1992;153:81
81. Hengartner A, Luzzati AL and Schreier M. Fusion of in vitro immunized lymphoid cells with X63Ag8. Curt Top Microbiol Immunol 1978; 81:92
82．金伯泉主编．细胞和分子免疫学．西安，世界图书出版公司1995年：118
83. Lo MS, Tsong TY,Conrad MK et al. Monoclonal antibody production by receptor-mediated electrically induced cell fusion. Nature 1984; 310:792
84. Vitetta ES and Uhr JW. Monoclonal an tibodies as agonists:an expanded role for their use in cancer therapy. Cancer Res 1994;54:5301
85. Riechmann L and Clark M. Reshaping human antibodies for therapy. Nature 1988;332:323
86. Chiswell DJ and McCafferty J. Phage antibodies: will now "Coliclonal" antibodies replace monoclonal antibodies? TIBTECH 1992; 10:80
87. Maclennan I. The center of hypermutation. Nature 1991;354:352
88. Ward ES, Gussow D, Griffiths AD et al. Binding activities of a repertoire of single Ig variable domain secreted from E. coli. Nature 1989;341:544
89. Pluckthun A.Mono-and bivalent antibody fragment produced in E. coli:engineering, folding and antigen binding. Immunol Rev 1992; 130:151
90. Barbas CF, Bain JD, Hoekstra DM et al.Semisynthethetic combinatorial antibody libraries:chemical solution to the diversity problem. PNAS 1992;89:4457
91. Tinter G, and Griffithe AD. Making antibodies by phage display technology. Ann Rev Immunol 1994;12:433
92．陈竞华，王琰，刘群英等。人源性噬菌体抗体库的构建及HbsAg单抗的筛选。中华微生物学和免疫学杂志，1995；15：158
93. Ali SY, Anderson HC,Sajdera SW.Enzymic and electron-microscopic analysis of extracellular matrix vesicle assiciated calcification in carting. Biochem J. 1997,132;56
94．陈筠．牙髓碱性磷酸酶的提纯、cDNA克隆和氢氧化钙对其活性、表达的影响：[学位论文]。第四军医大学口腔医学院，1991．9
95．郝建军 激素和生长因子与牙乳头细胞，牙髓细胞，牙胚分化和矿化关系的体外实验研究 第四军医大学 博士学位论文 1997 5
96. Hao J, Shi S,Niu Z et al.Mineralized nodule formation by human dental papilla cells in culture. Eur J Oral Sci 1997 Aug; 105(4): 318-24
97. Goseki M, Oida S, Nifuji A et al Properties of alkaline phosphatase of the human dental pulp. J Dent Res. 1990, 69:909
98．徐志凯 实用单克隆抗体技术第1版 陕西科学技术出版社 1992
99. Laboux O, Menanteau J, Pelhate S et al. Development of a monoclonai antibody against dentin phosphophoryn: a tool to study odontoblastic activity. Hybridoma. 1994; 13(2):143-146
100．陶义询 主编 免疫学的免疫学检验 第二版 人民卫生出版社 1997
101. J.D Beatty, Barbara G, William G et al. Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. Journal of immunological methods. 1987;100(1987): 173-179
102. Kulkarni GV, Jee SW, Thotakura S, et al Differential expresstion of DMP1,2 and 3transcripts in carly stages of tooth development. In Goldberg M, Robinson C, Boskey AL, eds. Proceeding 6th International conference on the Chemistry and Biology of Minerallized Tissues. Rosemont, IL:American Academy of Orthopaedic Surgeons. 2000; 161-195